There is an increasing number of cases and spread of Lyme and other tick-borne diseases in the United States. The same tick that can transmit the agent of Lyme disease, Borrelia burgdorferi, can simultaneously transmit other pathogens such as Babesia, Anaplasma, and Rickettsia. This is of great concern to the public and their physicians. This concern is reflected in a greater demand for tests that can provide information as to what is in an individual tick and what can be transmitted. Current diagnostic testing methods for these disease causing pathogens are generally limited to a single microbe and/or unreliable. No test exists that can cover the breadth of pathogens transmitted by ticks. In our Phase I application, we achieved our aim, the proof of concept, that our methodology could detect multiple microbes and even in a single tick. That system employs broad-range PCR and mass spectroscopy (PCR/ESI-MS). The assay identified and speciated broad groups of bacteria, including known tick- borne bacteria and flaviviruses. The success of this, coupled with the increasing demand and increasing number of searches performed for Borrelia alone, is the basis for Phase 2 to concentrate on the development of a tick-borne pathogen detection assay using the high throughput PCR/ESI-MS. In Phase 1, the proof of principle of detection of pathogens was accomplished with 16 primer pairs in 16 wells of a 96 well kitted plate. In Phase 2, Goal 1 of this is to increase clinically useful throughput time and efficiency of the assay and consequently reduce costs. We will have a target goal of 8 wells (PCR reactions) by eliminating redundancy and multiplexing. Goal 2: Optimize and add to these, Babesia detection primers. Goal 3: Create a replenishable bank of nucleic acid extraction from infected ticks for future investigation of this type of assay as well as tick-human interface research. This bank will be of commercial value. Upon the completion of Phase2, we will be in a position to provide a tick-microbial diagnostic service for researchers, physicians, public health entities, private and individual citizen sectors. This will be an important tool in management of Lyme and related tick-borne diseases for improved medical treatment and public health surveillance. It will also be the foundation for doing the same in patient skin, blood, joint fluid, and cerebrospinal fluid. PUBLIC HEALTH RELEVANCE: The ability to accurately identify multiple pathogens in the ticks capable of causing Lyme disease and related disorders will be important in management strategy of patients exposed or bitten by ticks. It will help reduce rising costs to the patient and the public health system.